It will be appreciated by those of skill in the art that the process of obtaining governmental regulatory approval of a composition of matter for use as a therapeutic agent in both animal and human patients is an arduous and complex process. One particular aspect of the process pertains to proving and establishing the molecular structure of the chemical entity for which regulatory approval is sought. To establish the molecular structure of a candidate chemical entity to the satisfaction of regulatory approval bodies, it is critically important to have a highly purified reference standard compound against which lots or batches of the candidate compound are compared during the process of proving and establishing the structure.
Additionally, a highly purified reference standard for a compound is very important to quantification aspects of the compound. As is known to one of skill in the art, aspects of quantification include in vitro release and stability data. If a highly pure reference standard for a candidate compound is used, high quality data concerning stability and potency can be produced for submission to a regulatory body. The submission of such data ultimately leads to the development and distribution of better and safer products to the consumer. Thus, the provision of a highly purified reference standard is a key point in the regulatory approval process for candidate therapeutic compounds. Indeed, the highly purified reference standard is the standard to which all development activity materials are critically compared.
It will further be appreciated by one of skill in the art that it is difficult to provide highly pure reference standards for compounds initially isolated as bacterial fermentation products. First of all, the presence of contaminants in the bacterial fermentation product makes the basic structure of the chemical entity of interest difficult to ascertain. Moreover, once a chemical structure is determined, the presence of myriad polar and/or non-polar contaminants make it very difficult to purify the compound to a form suitable for use as a therapeutic agent. More particularly, it is very difficult to purify a reference standard quality compound for use in regulatory approval processes.
A compound of particular interest is ivermectin, a bacterial fermentation product having anti-parasitic activity and which is used in the treatment of heart worms. Ivermectin is typically prepared as a fermentation product from Streptomyces avermitilis coupled with a "semi-synthesis" chemical reaction. By "semi-synthesis" is meant synthetic transformations carried out on a previously naturally-formed structure. Ivermectin is included within the general class of fermentation product compounds known as avermectins. Despite the recognized utility of ivermectin in the treatment of heart worms, particularly in horses, regulatory approval processes are made difficult by the lack of a highly purified reference standard ivermectin compound.
The disclosures of all the following patents are incorporated herein by reference.
U.S. Pat. No. 4,429,042 issued to Albers-Schonberc et al. on Jan. 31, 1984 (assignee Merck & Co., Inc.) discloses the isolation of ivermectin from fermentation media. Purification is described as optionally accomplished by chromatographic techniques including normal phase column chromatography utilizing silica gel, aluminum oxide, dextran gels, and the like, coupled with elution with various solvents. High performance liquid chromatography (HPLC) is also described as an optional chromatographic detection method.
U.S. Pat. No. 4,333,925 issued to Buhs et al. on Jun. 8, 1982 (assignee Merck & Co., Inc.) discloses derivatives of ivermectin compounds which are purified from the livers of animals that have been administered ivermectin, as well as from in vitro incubations of such compounds with animal liver preparations. Isolation and purification of the derivatives are carried out using solvent extraction and chromatographic techniques, including HPLC.
U.S. Pat. No. 5,077,398 issued to Matthews on Dec. 31, 1991 (assignee Merck & Co., Inc.) discloses a process for the isolation and purification of avermectin compounds, particularly avermectin B1 and B2, which have significant anti-parasitic activity. Crystallization and re-crystallization techniques which facilitate the crystallization, isolation and purification of avermectin B1 and B2 are discussed. Difficulties associated with separating B1a/B1b and B2a/B2b homologs are noted.
What is needed is an improved method for purifying the bacterial fermentation product ivermectin which produces a highly purified product, yet is simple in operation and practice. Despite the above-described efforts, such a method is not currently available in the art.